Field of the Invention
The present invention relates to a method for ion exchange chromatography, and specifically ion exchange chromatography for preparative RNA transcript separations of long RNA transcripts.
Description of the Related Art
RNA transcripts have strong potential as therapeutics, but effective purification of these RNA transcripts for introduction into the body remains a problem. The current landscape for the preparative chromatographic purification of RNAs utilizes reversed phase high performance (or high pressure) liquid chromatography (RP-HPLC) performed with both porous and non-porous sorbents [1]. Use of RP-HPLC presents many issues regarding process scale up. Small-particle-sized sorbents referenced lend themselves to extremely high pressure processes unsuitable for large scale unit operations. Additionally, the use of organic/flammable solvents along with high pressure liquid handling equipment can be prohibitive for large scale manufacturing. These challenges include the introduction of USP class II residual solvents (acetonitrile and methanol) into the drug substance, the potential need for an explosion proof production facility, and the many obstacles presented by large scale, high pressure processes, including cost and availability. The addition of organic solvent during purification also requires significant additional downstream processing to ensure adequate removal of the solvent from the drug substance. Introduction of ion pair reagents (e.g., alkylammonium salts) is also problematic, since this produces salt form heterogeneity as these organic salts interact with the phosphate backbone. Additional unit operations are required to remove the ion pair reagents, obtain the desired salt form, and remove residual organic solvents. Furthermore, the binding capacity of the resins contained within in the prior art for preparative RNA purification has been shown to be less than 10 mg RNA/mL resin, and in some cases having extremely low binding capacities of less than 0.02 mg/mL, which is not viable for large scale manufacturing. [6][7] These low binding capacities lead to limited process productivity and throughput and can be cost prohibitive.
Previous work has also been performed using anion exchange chromatography as the method for preparative purification of RNA [2] [3]. While this has proven to work adequately for separations of short synthetic RNAs or RNA transcripts of 300 nucleotides in size or less, these methods have not been shown to work for longer RNAs or RNA transcripts of greater than 500 nucleotides in length. In addition, these methods use weak anion exchangers and operate under non-denaturing conditions. Purification techniques for longer RNAs and full length transcripts for use in therapeutics is desirable, preferably techniques that are scalable, reproducible, and thus usable for large scale manufacturing of therapeutics.